Measuring SERCA-mediated calcium uptake in mouse muscle homogenates

Geromella, Mia S.; Braun, Jessica; Fajardo, Val A.

doi:10.1016/j.xpro.2022.101987

Cited by 7 publications

(3 citation statements)

References 12 publications

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“…Calcium Uptake. ATP-dependent calcium uptake was measured in cardiac muscle homogenates with an Indo-1 calcium fluorophore-based assay as previously published [ 22 , 23 ]. Homogenates were added to a reaction buffer (200 mM KCl; 20 mM HEPES; 10 mM NaN 3 ; 5 M N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine; 15 mM MgCl 2 , pH 7.0) along with Indo-1 (4 M final concentration; 50041, Biotum, Fremont, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Perimenopause Decreases SERCA2a Activity in the Hearts of a Mouse Model of Ovarian Failure

Barry,

Rouhana,

Braun

et al. 2024

Biomolecules

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Risk of cardiovascular disease mortality rises in women after menopause. While increased cardiovascular risk is largely attributed to postmenopausal declines in estrogens, the molecular changes in the heart that contribute to risk are poorly understood. Disruptions in intracellular calcium handling develop in ovariectomized mice and have been implicated in cardiac dysfunction. Using a mouse model of menopause in which ovarian failure occurs over 120 days, we sought to determine if perimenopause impacted calcium removal mechanisms in the heart and identify the molecular mechanisms. Mice were injected with 4-vinylcyclohexene diepoxide (VCD) to induce ovarian failure over 120 days, mimicking perimenopause. Hearts were removed at 60 and 120 days after VCD injections, representing the middle and end of perimenopause. SERCA2a function was significantly diminished at the end of perimenopause. Neither SERCA2a nor phospholamban expression changed at either time point, but phospholamban phosphorylation at S16 and T17 was dynamically altered. Intrinsic SERCA inhibitors sarcolipin and myoregulin increased >4-fold at day 60, as did the native activator DWORF. At the end of perimenopause, sarcolipin and myoregulin returned to baseline levels while DWORF was significantly reduced below controls. Sodium–calcium exchanger expression was significantly increased at the end of perimenopause. These results show that the foundation for increased cardiovascular disease mortality develops in the heart during perimenopause and that regulators of calcium handling exhibit significant fluctuations over time. Understanding the temporal development of cardiovascular risk associated with menopause and the underlying mechanisms is critical to developing interventions that mitigate the rise in cardiovascular mortality that arises after menopause.

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Section: Methodsmentioning

confidence: 99%

Perimenopause Decreases SERCA2a Activity in the Hearts of a Mouse Model of Ovarian Failure

Barry,

Rouhana,

Braun

et al. 2024

Biomolecules

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“…SERCA function was assessed in diaphragm and cardiac muscle homogenates using an oxalate-supported Ca 2+ uptake fluorimetric assay and an ionophore (A23187, Sigma Aldrich)-supported spectrophotometric SERCA activity assay, both fitted onto a 96-well plate using an M2 MultiMode plate reader (Molecular Devices) as previously described 85,86 . For both Ca 2+ uptake and SERCA activity, rates were normalized to total protein obtained from a BCA assay.…”

Section: Star Methodsmentioning

confidence: 99%

“…MultiMode plate reader (Molecular Devices) as previously described 85,86 . For both Ca 2+ uptake and SERCA activity, rates were normalized to total protein obtained from a BCA assay.…”

Section: Serca Functionmentioning

confidence: 99%

GSK3 inhibition improves skeletal muscle function and whole-body metabolism in the severe DBA/2Jmdxmouse model

Hamstra

Whitley

Braun

et al. 2022

Preprint

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Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder that leads to early mortality. We examined the pathogenic contribution of glycogen synthase kinase 3 (GSK3) to DMD using the mdx model. GSK3 is a serine/threonine kinase that has been implicated in other muscular dystrophies and our initial results showed that overactivation of GSK3 may contribute to increased disease severity found in DBA/2J (D2) mdx mice vs C57BL/10 mdx mice. In support of this, treating D2 mdx mice with the GSK3 inhibitor, tideglusib (10 mg/kg/day), increased muscle mass, strength, and fatigue resistance. We also found elevated proportions of oxidative fibers and increased utrophin mRNA, while muscle necrosis and oxidative stress were reduced. Finally, young D2 mdx mice displayed early diastolic dysfunction, and this was blunted with tideglusib treatment, an effect attributed to lowered oxidative stress and fibrosis. This study highlights the therapeutic potential of tideglusib and GSK3 inhibition for DMD.

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